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     <h1 class="green">Our project</h1>
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          <h3 class="section-heading">Background</h3>
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          <p>Do you ever stop and reflect on the small choices you make in your everyday life and what consequences it might have on your surroundings? Many of the chores and tasks we do daily may contribute significantly to the ever growing microplastic littering in marine habitats. In fact, a single basket of laundry may release as much as 700 000 microplastic particles with each wash [1]. Meanwhile, practicing sports on an artificial grass field contributes to the cumulative release of several tons of microplastic debris every year [2]. Indeed, making a conscious effort in your daily life will have a direct impact on the future of Earth.</p>
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          <p>Microplastics are tiny fragments of plastics, less than five millimeters long, that tend to accumulate in the environment due to low degradability [3]. The origin of these fragments can be divided into two larger categories; primary and secondary sources [4]. Primary sources correspond to personal care products and deterioration of larger plastic objects. Examples include shower gels, wear of car tyres and synthetic textiles [5]. Secondary sources originate from slower mechanical or chemical weathering processes on plastics already disposed in nature, as observed in long-term UV degradation of larger plastic bodies [3].</p>
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            <p>While there exists only limited evidence supporting the toxicity of direct exposure to microplastics, such as shedding from fleece clothing, the potential risk to biota and humans increases drastically when the fragments reach larger bodies of water [6]. To the primary consumers, the small plastic pieces can look indistinguishable from food stuff, allowing microplastics to enter the food chain. The particles have shown a propensity of accumulating upwards and are known to cause blockage of the gastrointestinal tract and interference with the reproduction of higher organisms [7]. Another concern of water-bound microplastics lies in their affinity for hydrophobic compounds. Both heavy metals and persistent organic pollutants, two priority pollutants according to the EPA Clean Water Act, end up accumulating around them [6][8]. Since these compounds often have high bioavailability and are only bound to the plastic through weak association, they readily leach off the plastic and can cause adverse effects on the endocrine system and central nervous system [9] [10]. Similarly, plasticizers, or low-weight phthalates, which are common additives in commercial plastics that offer improved flexibility and ductility, have been known to cause hormonal disruption in humans and wildlife [11].</p>
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            <p>Surely, with extensive research corroborating the complications of microplastics, stringent laws and regulations ought to have been put in place to prohibit microplastic pollution - to protect the marine biodiversity and human health?</p>
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            <p>Well, not quite.</p>
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            <p>According to the United Nations Environment Programme, microplastics started to appear in in consumable goods about fifty years ago and up until a few years ago the consumer awareness of their potential ramifications was close to nonexistent. However, in the past few years, the consumer apathy with regards to the subject started to cease and political actions were finally put in place. In 2015, former President Obama signed the <em>Microbead-Free Waters Act of 2015</em>, consequently banning plastic additives in cosmetics and personal care products in the US [12] [13]. Some countries have since then followed suit, but far from the majority. Perhaps most notably, the European Union has not made any considerable strides toward the banning of microplastic products [11].</p>
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          <p>Recent discussions regarding the realization of the UN development goal number 14 [14] concerning the conservation and sustainable use of marine resources once again shed light on the matter and triggered a demand from consumers that retailers limit the amount of products containing microplastics. This caused a domino-effect in the entire supply chain that forced businesses to act to prevent them from losing their competitive edge on the market. Consequently, several retailers with significant market shares chose to stop handing out free plastic bags and selling personal care products containing microplastics [15] [16].</p>
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          <p>While the issue is being addressed and regulations are moving in the right direction, the process is slow and without a realistic end in sight. We, team iGEM Lund, have therefore chosen to devote this year to participate in the world-wide engagement against microplastic accumulation in the ocean, in line with the United Nations new framework for sustainable development.</p>
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          <h3 class="section-heading">How can synthetic biology aid?</h3>
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          <p>With some clever insight into the dynamics of synthetic biology, it can be harnessed to combat various aspect of marine debris. This has been recognized by previous iGEM teams throughout the years and the problem of microplastics in particular has been targeted multiple times, mainly through different methods of biotic degradation [17] [18] [19]. However, while bio-degradation would certainly be the preferred approach, as it eliminates the plastic once and for all, the process is very time-consuming and it does not compete with the abiotic degradation methods that exists today [20] [21]. With this in mind, we set out to apply our knowledge of synthetic biology to alleviate the current microplastic situation.</p>
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          <p>After dialogues with water sanitation experts, it was made apparent that not only does the Swedish government allocate very little resources to the sanitation of microplastics in water treatment plants, but that there exists no quick and easy way of even detecting whether sanitation might be necessary without the use of expensive laboratory equipment and arduous filtration processes. Currently, the most efficient method of assessing microplastic content is through sieving and several subsequent filtration and separation steps [22]. A simple tool to evaluate the presence of microplastics would therefore be very helpful in that regard. Consequently, <em>μSense</em> was born.</p>
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          <h3 class="section-heading">Our solution</h3>
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          <p>For this year’s iGEM competition, we have decided to engineer a simple, novel biosensor with the goal of determining the presence of microplastics in freshwater. The sensor will be realized by design and implementation of a genetic circuit into <em>Escherichia coli. </em>A logic AND-gate will be constructed using the expression of two heterologous sensory elements to detect the presence of microplastics. For more information, see <a href="/Team:Lund/Design">project design</a>.</p>
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          <h3 class="section-heading">References</h3>
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                [1] Laura Paddison (2016, September 27). “Single clothes wash may release 700,000 microplastic fibres, study finds”. <em>The Guardian</em>. Retrieved 2017-09-01.
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                [2] KIMO (2017, February 27). “Microplastic Pollution from Artificial Grass – A Field Guide”. <em>KIMO (Kommunenes Internasjonale Miljøorganisasjon)</em>. Retrieved 2017-09-01.
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                [3] GESAMP (2016). “Sources, fate and effects of microplastics in the marine environment: part two of a global assessment” (Kershaw, P.J., and Rochman, C.M., eds). (IMO/FAO/UNESCO-IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection). Rep. Stud. GESAMP No. 93, 220 p
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                [4] Eerkes-Medrano, D., Thompson, R. and Aldridge, D. (2015). Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. <em>Water Research</em>, 75, pp.63-82.
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                [5] Boucher, J. and Friot D. (2017). “Primary Microplastics in the Oceans: A Global Evaluation of Sources”. <em>Gland, Switzerland: IUCN</em>, pp.43.
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                [6] Wright, S. and Kelly, F. (2017). Plastic and Human Health: A Micro Issue?. <em>Environmental Science &amp; Technology</em>, 51(12), pp.6634-6647.
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                [7] Tanaka, K. and Takada, H. (2016). Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters. <em>Scientific Reports</em>, 6(1).
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                [8] Federal Security Agency (2002). <em>Federal Water Pollution Control Act</em>.
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                [9] Avio, C., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L. and Regoli, F. (2015). Pollutants bioavailability and toxicological risk from microplastics to marine mussels.<em> Environmental Pollution</em>, 198, pp.211-222.
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                [10] Anderson, J., Park, B. and Palance, V. (2016). Microplastics in aquatic environments: Implications for Canadian ecosystems. <em>Environmental Pollution</em>, 218, pp.269-280.
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                [11] Jansen, J., Veldhuis, F. and Schreuder, M. (2017). <em>Towards a European ban on microbeads - Leiden Law Blog</em>. [online] Leidenlawblog.nl. [Accessed 1 Sep. 2017].
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                [12] 114th United States Congress (2015). An act to amend the Federal Food, Drug, and Cosmetic Act to prohibit the manufacture and introduction or delivery for introduction into interstate commerce of rinse-off cosmetics containing intentionally-added plastic microbeads.
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                [13] The National Oceanic and Atmospheric Administration (2017). What are microplastics?. [online] [Accessed 1 Sep. 2017].
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                [14] United Nations (2015). Transforming our world: the 2030 Agenda for Sustainable Development. [online] [Accessed 1 Sep. 2017]
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                [15] United Nations (2017). Goal 14 : Sustainable Development Knowledge Platform. [online] [Accessed 1 Sep. 2017].
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                [16] Lagercrantz, S. (2016). Apotea städar bort miljöbov<em>.</em> [online]<em> Dagens Medicin.</em> [Accessed 1 Sep. 2017].
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                [17] iGEM team University College London 2012 (2012). Welcome to Plastic Republic. [online] [Accessed 1 Sep. 2017].
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                [18] iGEM team Berlin 2015 (2015). Enzymatic Flagellulose<em>. </em>[online] [Accessed 1 Sep. 2017].
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                [19] iGEM team Virgin 2014 (2015). NyGone - a microplastic biofilter<em>. </em>[online] [Accessed 1 Sep. 2017].
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                [20] Ghosh, S., Pal, S. and Ray, S. (2013). Study of microbes having potentiality for biodegradation of plastics.<em> Environmental Science and Pollution Research</em>, 20(7), pp.4339-4355.
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                [21] Gewert, B., Plassmann, M. and MacLeod, M. (2015). Pathways for degradation of plastic polymers floating in the marine environment.<em> Environ. Sci.: Processes Impacts</em>, 17(9), pp.1513-1521.
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                [22] Masura, J., et al. (2015). Laboratory methods for the analysis of microplastics in themarineenvironment: recommendations for quantifying synthetic particles in watersand sediments. NOAA Technical Memorandum NOS-OR&amp;R-48
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    <h5>Hey there!</h5>
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     <p>We’re really glad that you would like to learn more about our iGEM-project for 2017! But first, could I trouble you with a question?</p>
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    <p>Do you every stop to think about the choices you make in your everyday life? Today I woke up, lets confess – after several rounds of snoozing – and brushed my teeth, did a load of laundry and hurried away to the obligations of today, there among my soccer practice at six o’clock. Not once would I have stopped to consider the simple formula of cause and effect in these everyday activities of mine, if it wasn’t for being a part of this iGEM-project, because who would have thought that choices made during a completely normal day could lead to environment destruction and the suffering of animals?  Not me! But the toothpaste I used, could have been a glittering one – a glitter that arises from the addition of microplastics in it, the load of laundry I did could have contained clothing in fabrics that caused release of about 700 000 microplastic particles [1] with each cycle in the washing machine and finally, maybe ran I – during my soccer practice – on a field made up of artificial grass leading to the release of about 1000 tonnes [2] of pollution particles discharged into the environment. Imagine the impact you can have on the Earths future; you are part of deciding its faith!</p>
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    <p>What we – and other people – refer to when talking about microplastics throughout this homepage, are tiny pieces of plastic – less than five millimeters long – that can be found in many health and beauty products such as hair products and toothpastes, or arise from larger plastic debris that are degraded into these tiny pieces by for instance the UV-light during a sunny day. These particles are not, to our knowledge, harmful for human health but the origin of the problem arises when we wash these off our skin or clothes. Because when microplastics find their way to the water treatment plants, the filtration systems used there are currently not capable of catching these particles, they are just too small which means that they travel on and eventually end up in our marine environment instead – the ocean, the home of about 230 000 living organisms [3].</p>
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     <p>To plankton and fishes the microplastics look like food and they are ingested by a variety of aquatic organisms, leading to their entering in the food chain. A food chain that also we humans are part of which means that microplastics in the end can end up in our own tummies. These deceitful tiny pieces of plastic can interfere with digestion and reproduction in the aquatic species and may cause physical harm, but that is not the only problem [4]. They can also leach chemicals such as plasticizers – which is a compound added to give a plastic material improved flexibility and reduce brittleness – and attract toxic chemicals such as persistent organic pollutants (POPs) – which is an organic compound that is resistant to degradation and therefore could accumulate to a potentially toxic level.</p>
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     <p>One may think that this is a recent problem but can you remember a time when your tooth paste was not glittering? According to the United Nations environment Programme, microplastics started to appear in these kinds of products about fifty years ago and just five years back this issue was still not raised and consumer awareness was low. Although in 2015, former President Obama signed the Microbead-Free Waters Act of 2015 [5] and banned plastic additives in cosmetics and personal care products. [6] Also, recent discussions regarding the realization of the UN development goal number 14 concerning the conservation and sustainable use of marine resources have shed light on the matter and during the last year several companies have taken a stand by for instance not giving out plastic bags for free in the shopping malls [7] and avoid selling products containing microplastics [8].</p>
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     <p>We, iGEM Team Lund, therefore chose, in line with the UN's new framework for sustainable development, to devote this year to the increasingly aware issue of microplastic accumulation in the ocean.</p>
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    <p>Our project focuses on designing a way to determine the presence of microplastics in water by *drum roll* implementing a genetic circuit in E. coli and utilize a logic AND-gate to create a biosensor for indirect detection of microplastics using two different molecules – the plasticizer and POPs mentioned earlier and often associated with microplastics.</p>
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     <p>So, to break it down, E. coli is the, probably, most common lab bacteria and due to that; it is also easier to work with since a lot is known regarding it. For E. coli to live its life, it has – just like all other life on earth – a genetic code, in the form of DNA, which is transcribed to RNA and then translated into proteins that are useful for the organism itself. One such protein is Green Fluorescent Protein (GFP) which, like its name reveals, fluoresces green when expressed and therefore enables an easy way to detect and measure the amount of expression of that particular protein. But for a protein to be expressed, the DNA has to contain at least 4 things; a promotor – which marks where the gene should start being transcribed, a UTR (ribosome binding site) to where the ribosome can attach and do its translation into protein, your favorite gene – encoding whatever you would like to express, and a terminator – which marks where everything should end. Also if your gene is added to the bacterium in form of a plasmid, a plasmid backbone is also needed which contains, amongst other things, antibiotic resistance that gives an advantage to the bacterium which picks up the plasmid since the cells would not be able to survive the antibiotic we will be adding unless they have this plasmid. A genetic circuit is basically inserting DNA into a cell in order for it to carry out logical functions similar to what can be done in electronic circuits – and it is commonly used within synthetic biology. In this project, the gene of interest is GFP, but it is split into three pieces and it will only glow if those pieces are connected again. The promotor that is initiating transcription of one of the GFP-parts are only activated in the presence of POP, if POP is not existing in you water sample – then the GFP-part is not obtained – but if POP does exist in you water sample, then it will form a complex with nahr and activate the transcription of one of the GFP parts. But one GFP-part is not enough to receive a glowing measureable signal. The other two GFP-parts needed is connected to the two ends of hER-alpha (which is the alpha version of the estrogen receptor) and this protein is always expressed in this biosensor, but since the two GFP-parts are on either end of it, they will only be close enough to merge if phthalates are present in your water sample. If there are no phthalates in your water sample – nothing happens and no fluorescence is obtained, but if there is phthalates in your water sample then the hER-alpha receptor will bind them and go through a conformational change which causes the two parts of GFP to be close to enough to each other to work as GFP. Well, almost – because we would of course also need the third part of GFP that was expressed if POP was present in you sample. Think of it like playing hockey – if three persons wants to play hockey (kind of like our three GFP-parts wants to glow), but they only bring the hockey stick or they only bring the puck – it will be a bit hard to play… But if they bring both the hockey stick and the puck – now it’s looking to be a great afternoon! Same applies for the GFP-parts, they will only be able to fluoresce if all three of them are connected, which they can only be if both POPs and phthalates are present in your water sample, which these molecules likely are… if your water sample contains microplastics! So now that you understand this, that all three parts must be present, then you understand how a logic AND-gate work! And most likely, you’ll also very soon be able to rock in synthetic biology!</p>
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        <a href="https://www.theguardian.com/science/2016/sep/27/washing-clothes-releases-water-polluting-fibres-study-finds">
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          http://www.kimointernational.org/news/microplastic-pollution-from-artificial-grass-a-field-guide/
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          https://www.newscientist.com/article/dn14206-how-many-species-live-in-the-sea/
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Latest revision as of 00:38, 2 November 2017

Background

Do you ever stop and reflect on the small choices you make in your everyday life and what consequences it might have on your surroundings? Many of the chores and tasks we do daily may contribute significantly to the ever growing microplastic littering in marine habitats. In fact, a single basket of laundry may release as much as 700 000 microplastic particles with each wash [1]. Meanwhile, practicing sports on an artificial grass field contributes to the cumulative release of several tons of microplastic debris every year [2]. Indeed, making a conscious effort in your daily life will have a direct impact on the future of Earth.

Microplastics are tiny fragments of plastics, less than five millimeters long, that tend to accumulate in the environment due to low degradability [3]. The origin of these fragments can be divided into two larger categories; primary and secondary sources [4]. Primary sources correspond to personal care products and deterioration of larger plastic objects. Examples include shower gels, wear of car tyres and synthetic textiles [5]. Secondary sources originate from slower mechanical or chemical weathering processes on plastics already disposed in nature, as observed in long-term UV degradation of larger plastic bodies [3].

While there exists only limited evidence supporting the toxicity of direct exposure to microplastics, such as shedding from fleece clothing, the potential risk to biota and humans increases drastically when the fragments reach larger bodies of water [6]. To the primary consumers, the small plastic pieces can look indistinguishable from food stuff, allowing microplastics to enter the food chain. The particles have shown a propensity of accumulating upwards and are known to cause blockage of the gastrointestinal tract and interference with the reproduction of higher organisms [7]. Another concern of water-bound microplastics lies in their affinity for hydrophobic compounds. Both heavy metals and persistent organic pollutants, two priority pollutants according to the EPA Clean Water Act, end up accumulating around them [6][8]. Since these compounds often have high bioavailability and are only bound to the plastic through weak association, they readily leach off the plastic and can cause adverse effects on the endocrine system and central nervous system [9] [10]. Similarly, plasticizers, or low-weight phthalates, which are common additives in commercial plastics that offer improved flexibility and ductility, have been known to cause hormonal disruption in humans and wildlife [11].

Surely, with extensive research corroborating the complications of microplastics, stringent laws and regulations ought to have been put in place to prohibit microplastic pollution - to protect the marine biodiversity and human health?

Well, not quite.

According to the United Nations Environment Programme, microplastics started to appear in in consumable goods about fifty years ago and up until a few years ago the consumer awareness of their potential ramifications was close to nonexistent. However, in the past few years, the consumer apathy with regards to the subject started to cease and political actions were finally put in place. In 2015, former President Obama signed the Microbead-Free Waters Act of 2015, consequently banning plastic additives in cosmetics and personal care products in the US [12] [13]. Some countries have since then followed suit, but far from the majority. Perhaps most notably, the European Union has not made any considerable strides toward the banning of microplastic products [11].

Recent discussions regarding the realization of the UN development goal number 14 [14] concerning the conservation and sustainable use of marine resources once again shed light on the matter and triggered a demand from consumers that retailers limit the amount of products containing microplastics. This caused a domino-effect in the entire supply chain that forced businesses to act to prevent them from losing their competitive edge on the market. Consequently, several retailers with significant market shares chose to stop handing out free plastic bags and selling personal care products containing microplastics [15] [16].

While the issue is being addressed and regulations are moving in the right direction, the process is slow and without a realistic end in sight. We, team iGEM Lund, have therefore chosen to devote this year to participate in the world-wide engagement against microplastic accumulation in the ocean, in line with the United Nations new framework for sustainable development.

How can synthetic biology aid?

With some clever insight into the dynamics of synthetic biology, it can be harnessed to combat various aspect of marine debris. This has been recognized by previous iGEM teams throughout the years and the problem of microplastics in particular has been targeted multiple times, mainly through different methods of biotic degradation [17] [18] [19]. However, while bio-degradation would certainly be the preferred approach, as it eliminates the plastic once and for all, the process is very time-consuming and it does not compete with the abiotic degradation methods that exists today [20] [21]. With this in mind, we set out to apply our knowledge of synthetic biology to alleviate the current microplastic situation.

After dialogues with water sanitation experts, it was made apparent that not only does the Swedish government allocate very little resources to the sanitation of microplastics in water treatment plants, but that there exists no quick and easy way of even detecting whether sanitation might be necessary without the use of expensive laboratory equipment and arduous filtration processes. Currently, the most efficient method of assessing microplastic content is through sieving and several subsequent filtration and separation steps [22]. A simple tool to evaluate the presence of microplastics would therefore be very helpful in that regard. Consequently, μSense was born.

Our solution

For this year’s iGEM competition, we have decided to engineer a simple, novel biosensor with the goal of determining the presence of microplastics in freshwater. The sensor will be realized by design and implementation of a genetic circuit into Escherichia coli. A logic AND-gate will be constructed using the expression of two heterologous sensory elements to detect the presence of microplastics. For more information, see project design.

References

  1. [1] Laura Paddison (2016, September 27). “Single clothes wash may release 700,000 microplastic fibres, study finds”. The Guardian. Retrieved 2017-09-01.
  2. [2] KIMO (2017, February 27). “Microplastic Pollution from Artificial Grass – A Field Guide”. KIMO (Kommunenes Internasjonale Miljøorganisasjon). Retrieved 2017-09-01.
  3. [3] GESAMP (2016). “Sources, fate and effects of microplastics in the marine environment: part two of a global assessment” (Kershaw, P.J., and Rochman, C.M., eds). (IMO/FAO/UNESCO-IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection). Rep. Stud. GESAMP No. 93, 220 p
  4. [4] Eerkes-Medrano, D., Thompson, R. and Aldridge, D. (2015). Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. Water Research, 75, pp.63-82.
  5. [5] Boucher, J. and Friot D. (2017). “Primary Microplastics in the Oceans: A Global Evaluation of Sources”. Gland, Switzerland: IUCN, pp.43.
  6. [6] Wright, S. and Kelly, F. (2017). Plastic and Human Health: A Micro Issue?. Environmental Science & Technology, 51(12), pp.6634-6647.
  7. [7] Tanaka, K. and Takada, H. (2016). Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters. Scientific Reports, 6(1).
  8. [8] Federal Security Agency (2002). Federal Water Pollution Control Act.
  9. [9] Avio, C., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L. and Regoli, F. (2015). Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environmental Pollution, 198, pp.211-222.
  10. [10] Anderson, J., Park, B. and Palance, V. (2016). Microplastics in aquatic environments: Implications for Canadian ecosystems. Environmental Pollution, 218, pp.269-280.
  11. [11] Jansen, J., Veldhuis, F. and Schreuder, M. (2017). Towards a European ban on microbeads - Leiden Law Blog. [online] Leidenlawblog.nl. [Accessed 1 Sep. 2017].
  12. [12] 114th United States Congress (2015). An act to amend the Federal Food, Drug, and Cosmetic Act to prohibit the manufacture and introduction or delivery for introduction into interstate commerce of rinse-off cosmetics containing intentionally-added plastic microbeads.
  13. [13] The National Oceanic and Atmospheric Administration (2017). What are microplastics?. [online] [Accessed 1 Sep. 2017].
  14. [14] United Nations (2015). Transforming our world: the 2030 Agenda for Sustainable Development. [online] [Accessed 1 Sep. 2017]
  15. [15] United Nations (2017). Goal 14 : Sustainable Development Knowledge Platform. [online] [Accessed 1 Sep. 2017].
  16. [16] Lagercrantz, S. (2016). Apotea städar bort miljöbov. [online] Dagens Medicin. [Accessed 1 Sep. 2017].
  17. [17] iGEM team University College London 2012 (2012). Welcome to Plastic Republic. [online] [Accessed 1 Sep. 2017].
  18. [18] iGEM team Berlin 2015 (2015). Enzymatic Flagellulose. [online] [Accessed 1 Sep. 2017].
  19. [19] iGEM team Virgin 2014 (2015). NyGone - a microplastic biofilter. [online] [Accessed 1 Sep. 2017].
  20. [20] Ghosh, S., Pal, S. and Ray, S. (2013). Study of microbes having potentiality for biodegradation of plastics. Environmental Science and Pollution Research, 20(7), pp.4339-4355.
  21. [21] Gewert, B., Plassmann, M. and MacLeod, M. (2015). Pathways for degradation of plastic polymers floating in the marine environment. Environ. Sci.: Processes Impacts, 17(9), pp.1513-1521.
  22. [22] Masura, J., et al. (2015). Laboratory methods for the analysis of microplastics in themarineenvironment: recommendations for quantifying synthetic particles in watersand sediments. NOAA Technical Memorandum NOS-OR&R-48